Following the introduction of polysaccharide vaccines against Meningococcus A, C, Y and W135 and the conjugate vaccine against Haemophilus influenzae type b, Meningococcus B (MenB) is the major cause of meningitis worldwide. In the United States alone, Group B Neisseria meningitidis cause 46% of total meningitis cases (ref. 1--Throughout this application, various references are referred to in parenthesis to more fully describe the state of the art to which this invention pertains. Full bibliographic information for each citation is found at the end of the specification, immediately preceding the claims. The disclosures of these references are hereby incorporated by reference into the present disclosure). Meningococcal meningitis occurs in both endemic and epidemic forms. Epidemic disease occurs in all parts of the world and incidences as high as 500 per 100,000 have been reported. Without antibiotic treatment the mortality is exceedingly high (85%) and even with this treatment it still remains at approximately 10%. In addition, patients cured by antibiotic therapy can still suffer serious and permanent neurologic deficiencies. These facts together with the emergence of sulfadiazine-resistant strains promoted the rapid development of a commercial vaccine (ref. 2).
Meningitidis is a gram-negative bacterium that has been classified serologically into groups A, B, C, 29e, W135, X, Y and Z. Additional groups H, I, and K were isolated in China (ref. 4) and group L was isolated in Canada (ref. 5). The grouping system is based on the capsular polysaccharides of the organism.
In contrast to the pneumococcal vaccine, the composition of the meningococcal polysaccharide vaccine has been greatly simplified by the fact that fewer polysaccharides are required. In fact groups A, B, and C are responsible for approximately 90% of cases of meningococcal meningitis. Success in the prevention of group A and C meningococcal meningitis was achieved using a bivalent polysaccharide vaccine (refs. 6 and 7). This vaccine became a commercial product and has been used successfully in the last decade in the prevention and arrest of major meningitis epidemics in many parts of the world. However, there has been a need to augment this vaccine because a significant proportion of cases of meningococcal meningitis are due to groups other than A and C. Group B is of particular epidemiologic importance, but groups Y and W135 are also significant (ref. 8). The inclusion of the group B polysaccharide in the vaccine remains a special problem. However, a tetravalent vaccine comprising groups A, C, W135, and Y is the currently used meningococcal meningitis vaccine.
The group B meningococcal polysaccharide is poorly immunogenic in man (ref. 9). Two major reasons have been proposed to account for this phenomenon. One is that the .alpha.-(2.fwdarw.8)-linked sialic acid homopolymer is rapidly depolymerized in human tissue because of the action of neuraminidase. The other is that Group B capsular polysaccharide (CPs) consist of poly N-acetylneuraminic acid (.alpha. 2-8 NeuNAc), .alpha. 2-8 NeuNAc is found as monomers and dimers on glycoproteins and gangliosides in adults and as polymers with at least eight repeating units in rat fetal and newborn tissues. Thus, the structure is recognized as "self" by the human immune system and in consequence, the production of antibody specific for this structure is suppressed and because of this molecular mimicry, a vaccine based on the native group B CPs could raise antibody directed against the .alpha. 2-8 NeuNAc moiety that might induce autoimmune disease.
Since the group B meningococcal capsular polysaccharide (CPs) is not immunogenic in humans, approaches have been pursued to increase its immunogenicity. One approach uses non covalent complexes of Group B CPs and outer membrane protein (OMPs) (ref. 10). Such complexes are formed by hydrophobic interaction between hydrophobic regions on the OMPs and a diacyl glycerol group present on the reducing end of the CPs (ref. 11, 12). (The chemical structures of the meningococcal Group B and C capsular polysaccharides are shown in FIG. 3). Human volunteers were given two doses of the complex at 0 and 5 weeks. The antibody response to the this complex was initially encouraging, since most individuals responded with an increase in antibodies to Group B CPs (refs. 12, 13). However, the second dose resulted in little or no increase in antibody titres which declined over a period of 14 weeks. The antibodies with specificity for the Group B polysaccharide were limited to antibodies of the IgM class (ref. 10, 12) and were directed against determinants associated only with high molecular weight polysaccharides.
In a further attempt to improve the immunogenicity of Group B CPs, Jennings et al (ref. 14) prepared a Group B meningococcal-TT conjugate (GBMP-TT) by covalently linking the CPs to tetanus toxoid (TT) through its terminal non-reducing sialic acid using periodate oxidized CPs. This procedure, however, did not result in any significant enhancement in immunogenicity (ref. 14). The antibody response elicited in animals was directed primarily against the linkage point between the CPs and the protein (GBMP-TT).
A further approach to improve the immunogenicity of the Group B CPs involved the chemical modification of the polysaccharide. Jennings et al. (ref. 15) reported that N-acetyl group of Group B CPs could be selectively removed by the action of a strong base at elevated temperature. The acetyl groups were then replaced with N-propionyl groups by propionic anhydride treatment to produce poly N-propionylneuraminic acid (a (2-8) NeuPro). The N-propionylated CPs were first periodate oxidized with sodium periodate, and then coupled to TT in the presence of sodium cyanoborohydride to provide chemically modified GBMP-TT conjugate. When mice were immunized with this conjugate formulated in Freund's complete adjuvant (FCA), high levels of IgG antibody cross-reactive with the native Group B CPs were observed. The mouse anti-serum was found to be bactericidal for all Group B strains. However, further studies revealed that two populations of antibody with different specificity were found in the sera. One population reacted with purified Group B CPs and one did not. The antibodies that did not react with the isolated Group B CPs appeared to be responsible for bactericidal activity. These antibodies may be reacting with an epitope on the cell-associated CPs that was not present on the purified CP (ref. 16).
Neisseria meningitidis Group B is a serious pathogen for which no vaccine exists. It would be desirable to provide alternative conjugates comprising the capsular polysaccharide of N. meningitidis conjugated to a carrier as immunogenic compositions, including vaccines, and use as and for the generation of diagnostic reagents. In particular, the capsular polysaccharides of N. meningitidis contain multiple sialic derivatives that can be modified and used to attach carrier molecules thereto.